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Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells.

Ramnath RD, Sun J, Adhikari S, Bhatia M - J. Cell. Mol. Med. (2007 Nov-Dec)

Bottom Line: Moreover, substance P-induced ERK 1/2, JNK, NFkappaB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor.In addition, substance P-induced activation of ERK 112, JNK, NFkappaB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells.Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFkappaB and AP-1 signalling pathways in mouse pancreatic acini.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore.

ABSTRACT
Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFkappaB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lalpha (MIP-lalpha) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFkappaB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFkappaB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFkappaB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFkappaB and AP-1 signalling pathways in mouse pancreatic acini.

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Substance P stimulates ERK1/2 phosphorylation and NFκB activation in a time-dependent manner. Substance P induces a time-dependent phosphorylation of ERK1/2 which coincides with a time-dependent activation of NFκB. Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2, total ERK1/2 (B) Densitometric analysis of Western blot experiments from pancreatic acini. and (C) IκBα. In another experiment, the nuclear extract was used to isolate NFκB and ELISA was carried out to detect activation of (D) NFκB. The results are representative of three independent experiments. Results shown are the means + SE. # P≤0.05 when compared to 0 min control.
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fig01: Substance P stimulates ERK1/2 phosphorylation and NFκB activation in a time-dependent manner. Substance P induces a time-dependent phosphorylation of ERK1/2 which coincides with a time-dependent activation of NFκB. Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2, total ERK1/2 (B) Densitometric analysis of Western blot experiments from pancreatic acini. and (C) IκBα. In another experiment, the nuclear extract was used to isolate NFκB and ELISA was carried out to detect activation of (D) NFκB. The results are representative of three independent experiments. Results shown are the means + SE. # P≤0.05 when compared to 0 min control.

Mentions: To examine whether substance P causes ERK1/2 phosphorylation in pancreatic acini, mouse pancreatic acinar cells were treated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min. Cells were then lysed, and cell proteins were subjected to Western blot analysis using antibodies against both phospho-ERK1/2 and total ERK1/2. As shown in Figure 1A, substance P-induced phosphorylation of ERK1/2 in pancreatic acini that was evident at 3 min and increased in a time-dependent manner up to 120 min. Figure 1B, densitometric analysis of Western blot experiments revealed a significant increase in phosphorylation of ERK2 at all the above mentioned time points when compared to 0 min control. Phosphorylation of ERK1 was significantly higher at 45, 60 and 120 min when compared to 0 min control. The time-dependent increase in phosphorylation of ERK1/2 was in line with the time-dependent degradation of total IκBκ as shown in Figure 1C. In a similar experiment nuclear extract was used, instead of cell lysate, to detect NFκB activation by ELISA. As shown in Figure 1D, treatment with 1 μM substance P caused a time-dependent increase in NFκB activation as early as 3 min. The increase became significant at 10 min and reached maximum at 120 min after substance P treatment.


Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells.

Ramnath RD, Sun J, Adhikari S, Bhatia M - J. Cell. Mol. Med. (2007 Nov-Dec)

Substance P stimulates ERK1/2 phosphorylation and NFκB activation in a time-dependent manner. Substance P induces a time-dependent phosphorylation of ERK1/2 which coincides with a time-dependent activation of NFκB. Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2, total ERK1/2 (B) Densitometric analysis of Western blot experiments from pancreatic acini. and (C) IκBα. In another experiment, the nuclear extract was used to isolate NFκB and ELISA was carried out to detect activation of (D) NFκB. The results are representative of three independent experiments. Results shown are the means + SE. # P≤0.05 when compared to 0 min control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401295&req=5

fig01: Substance P stimulates ERK1/2 phosphorylation and NFκB activation in a time-dependent manner. Substance P induces a time-dependent phosphorylation of ERK1/2 which coincides with a time-dependent activation of NFκB. Freshly isolated pancreatic acini, obtained from three mice, were incubated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min at 37°C. In some experiments, cells were lysed and cell proteins were subjected to Western blot analysis using antibodies against (A) phospho-ERK1/2, total ERK1/2 (B) Densitometric analysis of Western blot experiments from pancreatic acini. and (C) IκBα. In another experiment, the nuclear extract was used to isolate NFκB and ELISA was carried out to detect activation of (D) NFκB. The results are representative of three independent experiments. Results shown are the means + SE. # P≤0.05 when compared to 0 min control.
Mentions: To examine whether substance P causes ERK1/2 phosphorylation in pancreatic acini, mouse pancreatic acinar cells were treated with 1 μM substance P for 0, 3, 5, 10, 15, 45, 60, 120 min. Cells were then lysed, and cell proteins were subjected to Western blot analysis using antibodies against both phospho-ERK1/2 and total ERK1/2. As shown in Figure 1A, substance P-induced phosphorylation of ERK1/2 in pancreatic acini that was evident at 3 min and increased in a time-dependent manner up to 120 min. Figure 1B, densitometric analysis of Western blot experiments revealed a significant increase in phosphorylation of ERK2 at all the above mentioned time points when compared to 0 min control. Phosphorylation of ERK1 was significantly higher at 45, 60 and 120 min when compared to 0 min control. The time-dependent increase in phosphorylation of ERK1/2 was in line with the time-dependent degradation of total IκBκ as shown in Figure 1C. In a similar experiment nuclear extract was used, instead of cell lysate, to detect NFκB activation by ELISA. As shown in Figure 1D, treatment with 1 μM substance P caused a time-dependent increase in NFκB activation as early as 3 min. The increase became significant at 10 min and reached maximum at 120 min after substance P treatment.

Bottom Line: Moreover, substance P-induced ERK 1/2, JNK, NFkappaB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor.In addition, substance P-induced activation of ERK 112, JNK, NFkappaB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells.Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFkappaB and AP-1 signalling pathways in mouse pancreatic acini.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore.

ABSTRACT
Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFkappaB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lalpha (MIP-lalpha) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFkappaB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFkappaB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFkappaB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFkappaB and AP-1 signalling pathways in mouse pancreatic acini.

Show MeSH
Related in: MedlinePlus